Fluorescence based sensors provide sensitive means of determining the presence of compounds of interest in a sample. Sensors may include a source of fluorescence signal, and a substrate for chemical or biocatalytic transformation. Various signal transduction methodologies can be use in measuring the response, where the sensor can produce a detectable change in fluorescence upon interacting with an analyte. Fluorescent sensors can provide desirable properties such as water solubility, low detection limits, and high selectivity for a desired analyte, where the analyte can be a small molecule, an enzyme, a catalytic metal, and the like.
Fluorescence quenching provides a useful tool in sensor design (Haugland, R. P. Handbook of Fluorescent Probes and Research Products, Ninth Edition; Molecular Probes: Eugene, Oreg., 2002). Over the past years, organic dyes have been used extensively, for example in FRET applications, but the requirements of FRET make matching a donor-acceptor pair difficult. (Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.; Kluwer Academic: New York, 1999).
As a result of the greatly expanding use of fluorescent labels in research and diagnostic applications, there is a corresponding increase in the need for very low background, for example as provided in a highly quenched fluorophore-quencher pair. The present invention addresses this need. It provides a class of fluorophores that can be exceptionally strongly quenched. Moreover, it provides pairings of quenchers with these fluorophores that yields this very low background fluorescence, and provides a specific structural design of sensor molecules that include these fluorophores and quenchers. Further, it provides uses for detection of a variety of analytes and conditions.
Publications
Wilson et al. (2007) J.A.C.S. 129:15426-15427 describe the efficient quenching of oligomeric fluorophores on a DNA backbone. Wilson et al. (2007) Tetrahedron 63(17):3427-3433 describe how fluorescent nucleobases can interact electronically to yield complexity in fluorescence emission. Cuppoletti et al. (2005) Bioconjug Chem. 16(3):528-34 describe oligomeric fluorescent labels for DNA. Kool et al. in U.S. Pat. No. 7,423,133 describes fluorescent glycosides and methods for their use. Each publication is herein specifically incorporated by reference.
Nagase et al. (1994) J.B.C. 33:20952-20957; and McIntyre et al. (2004) Biochem J. 377:617-628 teach fluorescently labeled protease sensing molecules with a fluorophore at one end and a quencher at the opposite end. Protease enzymes cleave the peptides, separating the quencher from the fluorophore. The increase in fluorescence is about 11 to about 17 fold, with substantial background fluorescence.
Jones et al., U.S. Patent Application 20080213747 describe fluorescent polymers and their use in superquenching-based bioassays. Jones et al. (2001) PNAS 98(26):14769-72 investigates fluorescence superquenching for polyelectrolytes consisting of cyanine dye pendant polylysines ranging in number of polymer repeat units (N(PRU)) from 1 to 900, both in solution and after adsorption onto silica nanoparticles. Lu et al. (2002) JACS 124:483-488 describe superquenching in cyanine pendant poly(l-lysine) dyes. Applications of fluorescent polymer superquenching to high throughput screening assays for protein kinases is discussed by Xia et al. (2004) Assay Drug Dev Technol. 2(2):183-92.